Cell Death: New Research Solutions for Apoptosis, Autophagy,

and Necrosis

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Cell Death: Key Points in History

1842: Carl Vogt identifies regulated cell death during vertebrate development

1964: The term programmed cell death first used

Publication on insect tissue development (Lockshin and Williams)

1972: The term apoptosis proposed (Kerr et al)

1976: Cell death recognized in Caenorhabditis elegans (Sulston)

1986: Bcl2 cloned (Cleary et al)

First component of the cell death system recognized

2002: Nobel Prize in Medicine for apoptosis

Brenner, Sulston, Horvitz

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Introduction: Cell Death

Definition: Cell death is a controlled mechanism necessary for development, immune regulation,
and homeostasis.

Other potential cell death mechanisms

Pyroptosis/pyronecrosis
Mitotic catastrophe
Excitotoxicity
Wallerian degeneration
Paraptosis
Entosis

Commonly accepted cell death mechanisms

Apoptosis
Autophagy
Necrosis
Cornification

Recognition of cell death

Cell morphology
Enzymology
Immunological/nonimmunological
Programmed/accidental
Physiological/pathological

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Apoptosis
Morphology
Membrane blebbing
Cell shrinkage
Chromatin condensation and
fragmentation
PS exposure
Phagocyte engulfment
Physiological functions
Embryonic Development
Cellular differentiation
Cellular damage
Infection
Pathophysiological functions
Cancer
Autoimmune diseases
Chronic inflammation

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Apoptosis Pathway

Duprez L (2009) Microbes & Infect. 11: 1050.

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Autophagy
Morphology
NO Chromatin condensation and
fragmentation
NO phagocyte engulfment
Cytoplasm vacuolization
Physiological functions
Antigen presentation
Unfolded protein response
Cellular survival
Caloric restriction
Pathophysiological functions
Lysosomal glycogen storage
diseases
Cancer
Neurodegenerative diseases

Levine B (2005) J. Clin. Invest. 115: 2679.

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Autophagy (Molecular Mechanism and Processes)

Duprez L (2009) Microbes & Infect. 11: 1050.

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Necrosis (Programmed Necrosis)

Morphology
Gain in cell volume
Organelle swelling
Plasma membrane rupture and
loss of contents
Physiological functions
Immune system stimulation
(inflammation)
Pathophysiological functions
Ischemia/reperfusion injury?
Neurodegenerative diseases?

http://www.pathology.med.ohio-state.edu/ext/MedEd/
Med2Visuals/Scripts/descall.idc?FolderNumber=10534

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Necrosis Pathway

Duprez L (2009) Microbes & Infect. 11: 1050.

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Cell Death Pathway Crosstalk

Zhivotovsky B (2010) Exp. Cell Res. 316: 1374.

Christofferson DE (2010) Current Opinion in Cell Bio. 22: 263.

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Cell Death Experimental Design & QIAGEN

Gene Expression
RT-PCR
Epigenetics
miRNA
DNA methylation
Histone modifications
Functional Studies
Reporter assays
siRNA/shRNA

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Principles of qRT-PCR: Overview

Real-Time PCR
Amplify and simultaneously quantify target DNA
Reverse Transcription Real-Time PCR
Amplify and simultaneously quantify mRNA

CT Values: Threshold Cycle

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Experimental Overview: Gene Expression Analysis

Stimulate Cells

Isolate RNA

RT-PCR

Data Analysis
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Apoptosis Studies: Gene Expression

Why is breast cancer resistant to apoptosis?

Experiment: TRAIL-induced apoptotic gene expression in normal breast cells vs. MDA-MB231 cells. (Human Apoptosis 384HT PCR Array; PAHS-3012)

Results: c-FLIP, STAT5A, and STAT5B are upregulated. STAT5 signaling is involved in the
development of resistance to TRAIL-induced apoptosis.

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Apoptosis Studies: Gene Expression

Why is breast cancer resistant to apoptosis?

Experiment: TRAIL-induced apoptotic gene expression in normal breast cells vs. MDA-MB231 cells. (Human Apoptosis 384HT PCR Array; PAHS-3012)

Results: c-FLIP, STAT5A, and STAT5B are upregulated. STAT5 signaling is involved in the
development of resistance to TRAIL-induced apoptosis.

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Epigenetics: Overview

Activated
Transcription Factors

miRNA
shRNA
siRNA

Protein A
NFB

p53

Transcription
Initiation Complex

mRNA A

Histones
p53 BS Me

Me

Me

NFB BS

DNA Methylation

Me Me

Ac

Structural Gene
Me

Histone-DNA
Interactions

Me Me

DNA Methylation

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miRNA Reagents from QIAGEN

miRNeasy
Isolation

Target Identification

miRNA
Studies

Expression
miScript miRNA PCR Arrays

3 UTR Reporters

Function
miRNA mimics & inhibitors

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Apoptosis Studies: miRNA Function

1) miRNA expression profiling in renal cancer cell lines (A498 and Caki2) shows significant
downregulation of hsa-miR-708.
2) Overexpression of miR-708 suppresses tumorigenicity and induces apoptosis.
3) Bioinformatic predictions of miR-708 targets include Survivin (BIRC5, a member of the
inhibitor of apoptosis family).
4) Survivin was confirmed as a miR-708 target via 3 UTR reporter assay.

Conclusion: miR-708 induces apoptosis via Survivin downregulation.

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Apoptosis Studies: miRNA Function

1) miRNA expression profiling in renal cancer cell lines (A498 and Caki2) shows significant
downregulation of hsa-miR-708.
2) Overexpression of miR-708 suppresses tumorigenicity and induces apoptosis.
3) Bioinformatic predictions of miR-708 targets include Survivin (BIRC5, a member of the
inhibitor of apoptosis family).
4) Survivin was confirmed as a miR-708 target via 3 UTR reporter assay.

Conclusion: miR-708 induces apoptosis via

Survivin downregulation.

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Apoptosis Studies: miRNA Function

1) miRNA expression profiling in renal cancer cell lines (A498 and Caki2) shows significant
downregulation of hsa-miR-708.
2) Overexpression of miR-708 suppresses tumorigenicity and induces apoptosis.
3) Bioinformatic predictions of miR-708 targets include Survivin (BIRC5, a member of the
inhibitor of apoptosis family).
4) Survivin was confirmed as a miR-708 target via 3 UTR reporter assay.

Conclusion: miR-708 induces apoptosis via Survivin downregulation.

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Epigenetics: Overview

Activated
Transcription Factors

miRNA
shRNA
siRNA

Protein A
NFB

p53

Transcription
Initiation Complex

mRNA A

Histones
p53 BS Me

Me

Me

NFB BS

DNA Methylation

Me Me

Ac

Structural Gene
Me

Histone-DNA
Interactions

Me Me

DNA Methylation

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Apoptosis: DNA Methylation and Histone Modification

Prolactinomas lacking dopamine receptors are resistant to chemotherapeutic treatmentinduced apoptosis. Why dont these tumors express dopamine receptors?

1) Measure methylation of the CpG island within the D2R gene.

Result: Tumors are hypermethylated, reducing gene expression

2) Measure D2R histone modifications.

Result: Tumors have high H3K27me3, a marker for gene silencing, and low H3K9ac, a
marker for active gene transcription.

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Apoptosis: DNA Methylation and Histone Modification

Prolactinomas lacking dopamine receptors are resistant to chemotherapeutic treatmentinduced apoptosis. Why dont these tumors express dopamine receptors?

1) Measure methylation of the CpG island within the D2R gene.

Result: Tumors are hypermethylated, reducing gene expression

2) Measure D2R histone modifications.

Result: Tumors have high H3K27me3, a marker for gene silencing, and low H3K9ac, a
marker for active gene transcription.

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Apoptosis: DNA Methylation and Histone Modification

Prolactinomas lacking dopamine receptors are resistant to chemotherapeutic treatmentinduced apoptosis. Why dont these tumors express dopamine receptors?

1) Measure methylation of the CpG island within the D2R gene.

Result: Tumors are hypermethylated, reducing gene expression

2) Measure D2R histone modifications.

Result: Tumors have high H3K27me3, a marker for gene silencing, and low H3K9ac, a
marker for active gene transcription.

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Cignal
Reporter
Assays:
Complete Solution
Reporter
Assays:
Overview
Transcriptional Regulatory Elements (TRE), which establish the
specificity of each reporter

TATA
box

Reporter Construct

GFP/firefly luciferase

Tandem repeats of

TRE

EGFP

TF

FL

Upstream Signaling
Events
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Autophagy: Reporter Assay

NOD2 and ATG16L1 are susceptibility genes in Crohns disease. Could their dysregulation
lead to autophagy disruption and the pathogenesis of Crohns?

Experiment: Does NOD2-dependent NFB signaling lead to autophagy? Yes. Are typical
Crohns NOD2 mutants pathological?

Conclusions: Crohns NOD2 mutants inhibit the autophagic response. NOD2 is also involved
in the antibacterial response, identifying additional potential mechanisms for Crohns
pathology.

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Autophagy: Reporter Assay

NOD2 and ATG16L1 are susceptibility genes in Crohns disease. Could their dysregulation
lead to autophagy disruption and the pathogenesis of Crohns?

Experiment: Does NOD2-dependent NFB signaling lead to autophagy? Yes. Are typical
Crohns NOD2 mutants pathological?

Conclusions: Crohns NOD2 mutants inhibit the autophagic response. NOD2 is also involved
in the antibacterial response, identifying additional potential mechanisms for Crohns
pathology.

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Autophagy: Reporter Assay

NOD2 and ATG16L1 are susceptibility genes in Crohns disease. Could their dysregulation
lead to autophagy disruption and the pathogenesis of Crohns?

Experiment: Does NOD2-dependent NFB signaling lead to autophagy? Yes. Are typical
Crohns NOD2 mutants pathological?

Conclusions: Crohns NOD2 mutants inhibit the autophagic response. NOD2 is also involved
in the antibacterial response, identifying additional potential mechanisms for Crohns
pathology.

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Epigenetics: Overview

Activated
Transcription Factors

miRNA
shRNA
siRNA

Protein A
NFB

p53

Transcription
Initiation Complex

mRNA A

Histones
p53 BS Me

Me

Me

NFB BS

DNA Methylation

Me Me

Ac

Structural Gene
Me

Histone-DNA
Interactions

Me Me

DNA Methylation

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Necrosis Studies: siRNA Knockdown

How does 24S-OHC (common brain-derived cholesterol metabolite) induce neuronal cell
death?

Experiment: Morphological studies of SH-SY5Y cells suggest necrosis as the primary form of
cell death. siRNA-mediated knockdown of RIPK1 tested this hypothesis.

Conclusion: SH-SY5Y cells treated with RIPK1 siRNA and 24S-OHC did not undergo cell
death, showing that 24S-OHC induces necrosis in neuronal cells.

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Necrosis Studies: siRNA Knockdown

How does 24S-OHC (common brain-derived cholesterol metabolite) induce neuronal cell
death?

Experiment: Morphological studies of SH-SY5Y cells suggest necrosis as the primary form of
cell death. siRNA-mediated knockdown of RIPK1 tested this hypothesis.

Conclusion: SH-SY5Y cells treated with RIPK1 siRNA and 24S-OHC did not undergo cell
death, showing that 24S-OHC induces necrosis in neuronal cells.

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Necrosis Studies: siRNA Knockdown

How does 24S-OHC (common brain-derived cholesterol metabolite) induce neuronal cell
death?

Experiment: Morphological studies of SH-SY5Y cells suggest necrosis as the primary form of
cell death. siRNA-mediated knockdown of RIPK1 tested this hypothesis.

Conclusion: SH-SY5Y cells treated with RIPK1 siRNA and 24S-OHC did not undergo cell
death, showing that 24S-OHC induces necrosis in neuronal cells.

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Apoptosis & Autophagy: Which Pathway to Choose?

Gene Expression Analysis
When does glucocorticoid stimulation induce autophagy or apoptosis in osteocytes?

Experiment: Treat mice with varying concentrations of prednisolone for 28 days. Tibial RNA
analyzed with Apoptosis and Autophagy RT2 Profiler PCR Arrays (PAMM-012 & PAMM-084)

Conclusion: Lower doses induced autophagy, and higher doses induced apoptosis. The
autophagic response may be induced during cellular stress to promote survival.

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Apoptosis & Autophagy: Which Pathway to Choose?

Gene Expression Analysis
When does glucocorticoid stimulation induce autophagy or apoptosis in osteocytes?

Experiment: Treat mice with varying concentrations of prednisolone for 28 days. Tibial RNA
analyzed with Apoptosis and Autophagy RT2 Profiler PCR Arrays (PAMM-012 & PAMM-084)

Conclusion: Lower doses induced autophagy, and higher doses induced apoptosis. The
autophagic response may be induced during cellular stress to promote survival.

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Sample & Assay Technologies

Apoptosis & Autophagy: Which Pathway to Choose?

Gene Expression Analysis
When does glucocorticoid stimulation induce autophagy or apoptosis in osteocytes?

Experiment: Treat mice with varying concentrations of prednisolone for 28 days. Tibial RNA
analyzed with Apoptosis and Autophagy RT2 Profiler PCR Arrays (PAMM-012 & PAMM-084)

Conclusion: Lower doses induced autophagy, and higher doses induced apoptosis. The
autophagic response may be induced during cellular stress to promote survival.

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Conclusions
Three major forms of cell death
Apoptosis
Autophagy
Necrosis
QIAGEN offers many methods to study these
cellular processes
Gene Expression
RT2 Profiler PCR Arrays
Epigenetics
miScript miRNA PCR Arrays
miScript miRNA System
EpiTect Methyl qPCR Arrays
Pyromark CpG Assays
EpiTect ChIP qPCR Arrays
Functional Studies
Cignal Reporter System
SureSilencing RNAi

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